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Application of compound for improving transplantation efficiency of human hematopoietic stem cells

A technology of hematopoietic stem cells and compounds, which is applied in the field of compounds to improve the efficiency of hematopoietic stem cell transplantation, can solve problems such as limited difficulty, and achieve the effects of improving migration ability, enhancing transplantation efficiency and therapeutic effect

Pending Publication Date: 2022-03-29
EDIGENE GUANGZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, some of the currently developed methods are not completely clear about the impact on other functions of HSC, or are limited by the difficulty of practical operation

Method used

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  • Application of compound for improving transplantation efficiency of human hematopoietic stem cells
  • Application of compound for improving transplantation efficiency of human hematopoietic stem cells
  • Application of compound for improving transplantation efficiency of human hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Separation of hematopoietic stem cells

[0134] The artificial hematopoietic stem cells used are mainly derived from umbilical cord blood. After the umbilical cord blood sample is retrieved from the hospital, it is rewarmed at room temperature for 0.5-1 hour. The blood sample is transferred from the blood collection bag to a 50ml centrifuge tube and mixed gently. Use a pipette gun to take 100 μl samples respectively for white blood cell count and loss of cells to detect CD34 positive ratio. The remaining blood samples were diluted 1:1 with normal saline containing 1% HAS and mixed gently by pipette. Put the mixed solution in a new 50ml centrifuge tube, add an appropriate amount of lymphocyte separation solution to the bottom of the tube, and centrifuge at 400g for 30 minutes. After centrifugation, take out the centrifuge tube slowly to keep the liquid level. Carefully absorb the buffy coat cells with a pipette, move to a new centrifuge tube, wash the cells w...

Embodiment 2

[0138] Embodiment 2 The cultivation of hematopoietic stem cells

[0139] First prepare the complete medium for hematopoietic stem cells, its components and dosage are as follows:

[0140] Table 2 The list of components used in the complete medium of hematopoietic stem cells

[0141] Dosage brand Item No. StemSpan SFMEII stem cell 09655 IL6 20ng / ml PeproTech 200-06 TPO 100ng / ml PeproTech 300-18-100 FLT3L 100ng / ml PeproTech AF-300-19-100 SCF 100ng / ml PeproTech 300-07-100

[0142] According to the counting result of embodiment 1, respectively divide 5x10 4 CD34+ cells were added to each well of a 24-well plate, and the cells were cultured for 24 hours with 1 ml of complete medium per well.

[0143] On the next day, the compound was dissolved in DMSO and mixed, and then diluted to a specific concentration with PBS (see Table 3). Add an equal volume of 10 μl of the diluted compound into the cell culture medi...

Embodiment 3

[0147] Example 3 Detection of CD184 protein expression

[0148] On the third day, prepare antibody premix: PBS+0.5% BSA solution premixes fluorescent antibodies BV510 conjugated anti-human CD34 (Biolegend clone 581), FITC conjugated anti-human CD90 (Biolegend, clone 5E10) and APCCy7 conjugated anti-human CD45RA (Biolegend, cloneHI100), add 50 μl / ml of each antibody, and store in the dark.

[0149] Collect the cells into a centrifuge tube, centrifuge at 400g for 5 minutes, remove the supernatant, add 50 μl of antibody premix (containing 2.5 μl of each antibody) to each sample, resuspend and mix well, and incubate at 4°C in the dark for 20 minutes. Add 1ml of PBS to each tube to wash, centrifuge at 400g for 5 minutes and resuspend with 50μl of PBS+0.5%BSA solution. Add 2.5 μl of PE-conjugated anti-CD184 antibody (Biolegend, clone 12G5) to each sample, incubate at 37°C for 30 minutes, wash, and use 7AAD staining to identify cell viability. Flow cytometer (Beckman Coulter CytoFL...

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Abstract

The invention discloses an application of a compound in improving the transplantation efficiency of human hematopoietic stem cells. The compound is selected from one or more than two of a microtubulin polymerization inhibitor, LND-212854, AZD0364, SCH772984, Pimsertib, Trametinib, IWR-1-endo, TTNPB, JNK-inhibitor IX and CHIR99021. The invention further discloses an application of the compound in improving the transplantation efficiency of the human hematopoietic stem cells. The compound provided by the invention can improve the expression of the hematopoietic stem cell surface protein CD184 through in-vitro short-time treatment without influencing the phenotype, motility rate and various characteristics of cells. Through the combination of the chemokine receptor CD184 expressed by the HSC and the chemokine ligand SDF1 in the bone marrow microenvironment, the migration ability of the HSC and the homing ability of the HSC in the bone marrow are improved, and the transplantation efficiency and the treatment effect of the HSC are enhanced.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to the use of a compound for improving the transplantation efficiency of human hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells (HSCs) are a type of stem cells that exist in bone marrow and blood, have the ability to self-renew, and differentiate into all types of blood cells. Based on its characteristics, hematopoietic stem cell transplantation (HSCT) is clinically considered to be the most effective and even the only treatment for various blood diseases and immune diseases. [0003] According to the source of HSC, HSCT can be divided into autologous stem cell transplantation and allogeneic stem cell transplantation. According to the type of HSC, it can be divided into cord blood (CB), recruited peripheral blood (Mobilized peripheral blood, mPB) and bone marrow (Bone marrow, BM) HSC. At present, CB-HSC has received more and more attention, bu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K31/475A61K31/165A61P35/00A61P35/02A61P37/02A61P3/00A61P7/00A61P7/06
CPCA61K45/00A61K31/475A61K31/165A61P35/00A61P35/02A61P37/02A61P3/00A61P7/00A61P7/06A61K35/28C12N5/0647A61K31/506A61K31/519A61K31/4985A61K31/44A61K31/192A61K31/381C12N2501/599A61K2300/00C12N2500/38C12N2501/155C12N2501/415C12N2501/727C12N2501/999
Inventor 方日国马奎莹李超袁鹏飞
Owner EDIGENE GUANGZHOU INC
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